Antinociceptive Actions of Botulinum Toxin A1 on Immunogenic Hypersensitivity in Temporomandibular Joint of Rats

Botulinum neurotoxin type A1 (BoNT-A) reduces the peripheral peptide and cytokine upregulation in rats with antigen-evoked persistent immunogenic hypersensitivity (PIH) of the temporomandibular joint (TMJ). Herein, we examined the effects of two preparations of BoNT-A, abobotulinumtoxinA (aboBoNT-A; Dysport) and onabotulinumtoxinA (onaBoNT-A; Botox), on spontaneous and evoked nociceptive behaviors, as well as on central neuronal and astroglial activation. The antigen-evoked PIH was induced in rats via repeated systemic and unilateral intra-articular (i.a.) injections of methylated bovine serum albumin (mBSA). Rats were subsequently injected with unilateral i.a. aboBoNT-A (14 U/kg), onaBoNT-A (7 U/kg), or the vehicle (saline). After i.a. treatments, spontaneous and mechanically evoked nocifensive behaviors were assessed before and after the low-dose i.a. formalin (0.5%) challenge. The central effects of BoNT-A were assessed by an immunohistochemical analysis of cleaved synaptosomal-associated protein 25 (cSNAP-25) presence, c-Fos, GFAP, and CGRP expression in the trigeminal nucleus caudalis (TNC). Both BoNT-A preparations similarly reduced the formalin-induced spontaneous pain-related behaviors and mechanical allodynia of the hypernociceptive rats. Likewise, their effects were associated with the central occurrence of cSNAP-25 and reduction of c-Fos and GFAP upregulation in the TNC. BoNT-A antinociceptive activity on the PIH is associated with the toxin axonal transport to trigeminal sensory areas and reduction of neuronal and glial activation in central nociceptive regions.

Introduction
Temporomandibular joint (TMJ) rheumatoid arthritis (RA) is a chronic inflammatory condition involving a range of local and systemic proinflammatory mediators. TMJ-RA produces structural and functional deterioration of the affected TMJ and associated tissues [1]. TMJ-related symptoms are present in almost 65% of RA patients and are typically bilateral [2,3]. Along with the impairment of normal functions of the jaw joint, the TMJ-RA is often associated with periods of alternating nonpainful and painful phases [1]. Active painful phases are usually provoked by an otherwise nonpainful stimulus (e.g., mandibular functional movements: speaking, eating, etc.), with a negative impact on the patient's quality of life [4,5]. Notwithstanding, treatments with nonsteroidal anti-inflammatory drugs (NSAIDs) and local anesthetic into the TMJ compartment have proven to relieve RA pain just temporarily [6]. Animal models of RA-related pain, including TMJ-RA, have been proposed with the goal to improve the understanding of its etiology and develop new treatment options [7][8][9]. When used as an antigen, systemic and unilateral intra-TMJ administrations of methylated bovine serum albumin (mBSA) in rats result in the development of persistent immunogenic hypersensitivity (PIH) and inflammatory changes of the exposed TMJ ( Figure 1) [10,11]. Interestingly, even though this model resembles human TMJ-RA, it has a monoarthritic (single joint) presentation, since it is induced locally inside the mBSA-stimulated joint [8,10,11]. The hypersensitivity appears as an exaggerated pain-related response (facial grooming and head flinching) to a normally nonpainful chemical stimulation of the TMJ, such as a low-dose (0.5%) formalin challenge [8,12]. This arthritic model produces a delayed-type hypersensitivity (viz., type IV hypersensitivity) and is characterized by central sensitization due to immune and neuronal cells activation, leading to the local release of a variety pronociceptive factors [10,11,13,14]. This fact increases the validity of the model to study the progression of pain on chronic inflammatory conditions and to test promising treatment options. typically bilateral [2,3]. Along with the impairment of normal functions of the jaw joint, the TMJ-RA is often associated with periods of alternating nonpainful and painful phases [1]. Active painful phases are usually provoked by an otherwise nonpainful stimulus (e.g., mandibular functional movements: speaking, eating, etc.), with a negative impact on the patient's quality of life [4,5]. Notwithstanding, treatments with nonsteroidal antiinflammatory drugs (NSAIDs) and local anesthetic into the TMJ compartment have proven to relieve RA pain just temporarily [6]. Animal models of RA-related pain, including TMJ-RA, have been proposed with the goal to improve the understanding of its etiology and develop new treatment options [7][8][9]. When used as an antigen, systemic and unilateral intra-TMJ administrations of methylated bovine serum albumin (mBSA) in rats result in the development of persistent immunogenic hypersensitivity (PIH) and inflammatory changes of the exposed TMJ ( Figure  1) [10,11]. Interestingly, even though this model resembles human TMJ-RA, it has a monoarthritic (single joint) presentation, since it is induced locally inside the mBSAstimulated joint [8,10,11]. The hypersensitivity appears as an exaggerated pain-related response (facial grooming and head flinching) to a normally nonpainful chemical stimulation of the TMJ, such as a low-dose (0.5%) formalin challenge [8,12]. This arthritic model produces a delayed-type hypersensitivity (viz., type IV hypersensitivity) and is characterized by central sensitization due to immune and neuronal cells activation, leading to the local release of a variety pronociceptive factors [10,11,13,14]. This fact increases the validity of the model to study the progression of pain on chronic inflammatory conditions and to test promising treatment options. In animals exposed to PIH and challenged with a low-dose formalin in the TMJ, we previously demonstrated that a treatment with an intra-TMJ injection of botulinum neurotoxin type A1 (BoNT-A), a potent neurotoxin naturally produced by Clostridium botulinum, reduced spontaneous behavioral facial grooming and head flinching responses 24h and 14 days after its administration [8]. Additionally, BoNT-A reduced the In animals exposed to PIH and challenged with a low-dose formalin in the TMJ, we previously demonstrated that a treatment with an intra-TMJ injection of botulinum neurotoxin type A1 (BoNT-A), a potent neurotoxin naturally produced by Clostridium botulinum, reduced spontaneous behavioral facial grooming and head flinching responses 24h and 14 days after its administration [8]. Additionally, BoNT-A reduced the upregulated levels of substance P and calcitonin gene-related peptide (CGRP) in the trigeminal ganglion and interleukin 1 beta (IL-1β) in the TMJ of exposed rats [8]. Despite the apparent association of BoNT-A antinociceptive action with the mentioned peripheral effects, numerous studies in different pain models have suggested a necessity for an axonal transport and a direct central analgesic action of the toxin [15,16]. Therefore, we examined the occurrence of cleaved SNAP-25 (cSNAP-25), along with nociceptive neuronal and astroglial activation, on the trigeminal brainstem sensory areas of rats induced to PIH and treated with BoNT-A. In addition, spontaneous and evoked nociceptive behaviors were also assessed, and the efficacy of two different commercially available pharmaceutical preparations of BoNT-A: abobotulinumtoxinA (aboBoNT-A) and onabotulinumtoxinA (onaBoNT-A) administered at equiefficatious doses were compared.

BoNT-A Effects on Rat Grimace Scale (RGS) and Spontaneous Nocifensive Responses (Flinching, Sctratching)
Previously in the PIH model, the number of spontaneous motor responses (flinches and scratching) after the formalin stimulation was shown to be reduced by onaBoNT-A [8]. Herein, we extended the measurement of spontaneous nociception by examining facial grimacing related to pain (RGS) and examined if the spontaneous nociceptive responses are present both before and after TMJ stimulation with low-dose formalin 0.5%). In the RGS test, we found a lack of painful facial expression (score = 0) and a low number of behavioral nocifensive responses in all groups at the pre-formalin assessments. Conversely, the postformalin assessments showed higher values of RGS (Figure 2A,B) and a significant increase in the nocifensive responses ( Figure 2C) on induced animals compared to noninduced.  (RGS) prior to and after low-dose (0.5%) formalin stimulation of the temporomandibular joint (TMJ) of hypernociceptive rats. (A) Representative photographs showing rat facial expression in noninduced + saline (noninduced), induced + saline (induced), and induced + abobotulinumtoxinA (aboBoNT-A) animals after formalin stimulation. In the photos, the best visible features of nociceptive grimacing are orbital tightening, as well as characteristic spread-out ear positioning in animals experiencing more intensive pain (induced + saline vs. induced + onaBoNT/A), compared to no observable painful grimacing (noninduced + saline). (B) Both BoNT-A pharmaceutical preparations reduced the nociceptive facial grimacing evoked by 0.5% formalin, as assessed by RGS. No face grimacing was observed at the pre-formalin assessments. The individual RGS are calculated as the average of 4 distinctive facial features (orbital tightening, nose/cheek bulging, ear positioning, and whisker positioning) on a scale from 0 to 2. Mean ± SEM; +++ p < 0.001 vs. induced + saline (two-way repeated measures ANOVA followed by Bonferroni s post hoc test). (C) Both BoNT-A intra-articular (i.a.) treatments (14 U/kg aboBoNT-A or 7 U/kg onaBoNT-A) reduced the spontaneous nocifensive behaviors (total sum of the number of flinches-N, plus the duration of facial grooming and rubbing-s) evoked by low-dose (0.5%) i.a. formalin in rats exhibiting persistent immunogenic hypersensitivity compared to noninduced animals. N(animals/group) = 10. Mean ± SEM, *** p < 0.001 and ** p < 0.01 vs. noninduced + saline and +++ p < 0.001 vs. induced + saline (F 3,36 = 49.59, one-way ANOVA followed by Tukey's post hoc test).
Compared to induced animals treated with saline, both BoNT-A groups exhibited a significant reduction of the nocifensive responses triggered by formalin ( Figure 2C). Furthermore, both BoNT-A treatments resulted in a similar reduction (p < 0.01) of the RGS score after formalin injection ( Figure 2B).

Effects of BoNT-A on Mechanically-Evoked Responses at the TMJ Area
By employing the von Frey filaments, we examined the possible occurrence of facial mechanical allodynia over the skin covering the stimulated TMJ area both before and after the stimulation with 0.5% formalin. All animals showed a lack of allodynic responses at the pre-formalin evaluation. Following the formalin challenge, PIH-induced animals developed bilateral mechanical allodynia over the area of TMJ, measured with von Frey filaments ( Figure 3A,B). The after-formalin assessments showed that BoNT-A (aboBoNT-A and onaBoNT-A) reduced the mechanically evoked TMJ bilateral allodynia of the hypersensitive rats ( Figure 2A   . The values on the right y-axis represent the dose range of filaments used (in grams) on the corresponding logarithmic scale. N(animals/group) = 10; *** p < 0.001 and ** p < 0.01 vs. noninduced + saline and +++ p < 0.001 vs. induced + saline (two-way repeated measures ANOVA followed by Bonferroni's post hoc test).

Effect of BoNT-A on Neuronal and Astrocyte Activation in the TNC
We further examined the possible BoNT-A effects on neuronal and astrocyte activation associated with nociception in the trigeminal nucleus caudalis (TNC), the first-order The values were shown as the log-transformed data (log 10 (value in grams)). The values on the right y-axis represent the dose range of filaments used (in grams) on the corresponding logarithmic scale. N(animals/group) = 10; *** p < 0.001 and ** p < 0.01 vs. noninduced + saline and +++ p < 0.001 vs. induced + saline (two-way repeated measures ANOVA followed by Bonferroni's post hoc test).

Effect of BoNT-A on Neuronal and Astrocyte Activation in the TNC
We further examined the possible BoNT-A effects on neuronal and astrocyte activation associated with nociception in the trigeminal nucleus caudalis (TNC), the first-order pain processing sensory nucleus of the cranial area. We assessed the post-formalin neuronal activation of second-order nociceptive neurons in the TNC by employing c-Fos immunohistochemistry ( Figure 3A,B) and the counting of c-Fosexpressing neuronal profiles ( Figure 2C). After a normally nonpainful formalin injection, induced animals presented high levels of c-Fos-positive nuclei in both ipsilateral and contralateral TNC. Unilateral applications of BoNT-A (aboBoNT-A or onaBoNT-A) significantly decreased post-formalin c-Fos expression in both ipsilateral and contralateral TNC ( Figure 4B,C). Analysis of TNC showed an ipsilateral increase in astrocytic marker glial fibrillary acidic protein (GFAP) immunoreactivity on induced rats compared to noninduced animals ( Figure 5A). BoNT-A reduced the GFAP-immunoreactive area 14 days after treatment ( Figure 5B,C). The levels of calcitonin gene-related peptide (CGRP) expression were not increased in the hypernociceptive rats or altered by BoNT-A (aboBoNT-A or onaBoNT-A) treatments ( Figure 6).

Immunohistochemical Localization of cSNAP-25 in the Brain
Previously, we discovered that the BoNT/A antinociceptive action in the craniofacial area is associated with the occurrence of cleaved SNAP-25 (cSNAP-25) in the TNC following axonal transport [17]. Thus, we further examined if the antinociceptive actions of aboBoNT/A and onaBoNT/A are similarly associated with the direct toxin activity in the TNC. Fourteen days after BoNT-A (aboBoNT-A or onaBoNT-A) i.a. injections into the left TMJ, BoNT-A cSNAP-25-positive nerve fibers were found in the ipsilateral trigeminal sensory regions (TNC). Similar cSNAP-25 staining was observed in both aboBoNT-A and onaBoNT-A-treated animals. cSNAP-25 on the contralateral (nontreated) side was not observed (Figure 7).

Immunohistochemical Localization of cSNAP-25 in the Brain
Previously, we discovered that the BoNT/A antinociceptive action in the craniofacial area is associated with the occurrence of cleaved SNAP-25 (cSNAP-25) in the TNC following axonal transport [17]. Thus, we further examined if the antinociceptive actions of aboBoNT/A and onaBoNT/A are similarly associated with the direct toxin activity in the TNC. Fourteen days after BoNT-A (aboBoNT-A or onaBoNT-A) i.a. injections into the left TMJ, BoNT-A cSNAP-25-positive nerve fibers were found in the ipsilateral trigeminal sensory regions (TNC). Similar cSNAP-25 staining was observed in both aboBoNT-A and onaBoNT-A-treated animals. cSNAP-25 on the contralateral (nontreated) side was not observed (Figure 7).

Discussion
In rats induced to PIH of the TMJ, we demonstrated that the analgesic activity of BoNT-A, expressed as a reduction of spontaneous and evoked nocifensive behaviors after a low-dose formalin stimulation, is associated with a decrease of neuronal and astroglial activity in the TNC. Moreover, in the same arthritis model, we previously demonstrated that BoNT-A reduced the nocifensive behaviors, as well as the expression of proinflammatory mediators and peptides in the TMJ and trigeminal ganglion [8], in accordance with other studies [18][19][20]. Notwithstanding, a causal role between BoNT-A antinociceptive activity and its peripheral effects has not been established. Furthermore, it was suggested that the BoNT-A analgesic effect necessarily involves an axonal transport of the toxin to central nervous system (CNS), where it may interact with neuronal and/or non-neuronal cells [15,21].

Peripherally Injected BoNT-A Reduces Spontaneous and Mechanically Evoked Nocifensive Behaviors in Rats with PIH
Despite the existence of numerous tests for animals' behavior assessment [22], spontaneous behavioral assessments may be more relevant than stimulus-evoked assessments for future clinical translations, increasing the validity of the model [7]. However, the orofacial region remains an underrepresented area in spontaneous pain behavioral assessments. Persistent scratching and rubbing of the facial region after chemical or inflammatory stimulation (e.g., the orofacial formalin test) is used as a sign of pain in freely moving rodents [23,24]. For this reason, we also assessed the spontaneous nociceptive behaviors by employing RGS. Since pain is a well-characterized emotion based on the facial action

Discussion
In rats induced to PIH of the TMJ, we demonstrated that the analgesic activity of BoNT-A, expressed as a reduction of spontaneous and evoked nocifensive behaviors after a low-dose formalin stimulation, is associated with a decrease of neuronal and astroglial activity in the TNC. Moreover, in the same arthritis model, we previously demonstrated that BoNT-A reduced the nocifensive behaviors, as well as the expression of proinflammatory mediators and peptides in the TMJ and trigeminal ganglion [8], in accordance with other studies [18][19][20]. Notwithstanding, a causal role between BoNT-A antinociceptive activity and its peripheral effects has not been established. Furthermore, it was suggested that the BoNT-A analgesic effect necessarily involves an axonal transport of the toxin to central nervous system (CNS), where it may interact with neuronal and/or non-neuronal cells [15,21].

Peripherally Injected BoNT-A Reduces Spontaneous and Mechanically Evoked Nocifensive Behaviors in Rats with PIH
Despite the existence of numerous tests for animals' behavior assessment [22], spontaneous behavioral assessments may be more relevant than stimulus-evoked assessments for future clinical translations, increasing the validity of the model [7]. However, the orofacial region remains an underrepresented area in spontaneous pain behavioral assessments. Persistent scratching and rubbing of the facial region after chemical or inflammatory stimulation (e.g., the orofacial formalin test) is used as a sign of pain in freely moving rodents [23,24]. For this reason, we also assessed the spontaneous nociceptive behaviors by employing RGS. Since pain is a well-characterized emotion based on the facial action Toxins 2022, 14, 161 9 of 17 coding system [25], the RGS is capable of detecting spontaneous nociception [22] and measuring the analgesic efficacy of drugs [26].
The arthritic rats showed no visible signs of evoked or spontaneous pain prior to formalin TMJ stimulation. However, low-dose formalin (0.5%), known not to induce painrelated behaviors in normal animals [27], induced a significant increase of facial grimacing, head flinching, facial rubbing, and bilateral mechanical allodynia (Figures 2 and 3), in accordance with previous studies [8,28]. This suggests that the mBSA-induced monoarthritis is nonpainful per se; however, it induces a profound chemical hypersensitivity to normally nonpainful concentrations of inflammatory substances such as formalin [12]. This exacerbation of nociceptive responses by normally nonpainful chemical stimuli suggests a quick triggering of central sensitization processes [10].
BoNT-A decreased the flinching and rubbing nocifensive responses ( Figure 2C) and reduced the facial nociceptive grimacing of rats exposed to PIH in the TMJ. Importantly, the RGS was quantified at three different periods after formalin stimulation in the TMJ. Shortly after the formalin injection (0-5 min), BoNT-A was not effective in RGS reduction, similar to the observed lack of BoNT-A action on immediate formalin-evoked pain sensation (phase I of formalin test) [17,19]. However, at later time points (9-14 and 18-23 min), both BoNT-A groups significantly reduced the spontaneous pain grimacing score ( Figure 2B), demonstrating BoNT-A action in the tonic pain phase when central sensitization takes place.
In addition, we assessed the mechanical allodynia by von Frey monofilaments prior and after 0.5% formalin stimulation of the exposed TMJ. Allodynia over the TMJ area in PIH-induced rats was observed only after the 0.5% formalin challenge at both sides (ipsi-and contralateral TMJ). This bilateral allodynia was reduced by a unilateral BoNT-A treatment resembling the toxin's bilateral action on experimental trigeminal neuropathic pain [29] and polyneuropathic pain [16].

Effects of BoNT-A on Nociceptive Neuronal and Glial Activation
Apart from BoNT-A behavioral antinociceptive activity, we examined its action at the level of central sensory nociceptive nuclei by exploiting c-Fos activation as a reliable marker of neuronal activity [23,30]. Interestingly, we found an occurrence of c-Fos activated neurons in the ipsilateral and contralateral TNC after 0.5% formalin stimulation of the TMJ area (Figure 4), which was not observed in the orofacial or hind paw formalin tests [31][32][33]. The unilateral stimulation of mouse masseter with complete Freund's adjuvant (CFA) similarly induces bilateral expression of c-Fos in the TNC [23]. This suggests that pain in the TMJ region and related muscles may be associated with the bilateral activation of trigeminal nociception, similar to referred pain in humans [34,35]. All these results indicate that the pain triggered by formalin activates contralateral trigeminal nociceptive nuclei, explaining the occurrence of bilateral mechanical allodynia ( Figure 3A,B). Importantly, both BoNT-A preparations (aboBoNT-A and onaBoNT-A) injected into the ipsilateral TMJ reduced both bilateral neuronal activation and the bilateral allodynia in the TMJ area.
In addition, we examined the possible changes in the activation of glial cells in the CNS, since they are key to initiate and maintain central sensitization after peripheral tissue/nerve injury [10,36,37]. Astrocytes represent the majority of glial cells and play a significant role in the persistence of pain [38]. Both BoNT-A preparations reduced GFAP (astrocyte marker) immunoreactivity measured by surface area immunoreactivity and grey intensity in the TNC of arthritic rats ( Figure 5). An in vitro study showed a slight or no direct effect of BoNT-A on astrocytes [39]. Another in vivo study detected the presence of cSNAP-25 in spinal cord astrocytes of a mouse with induced neuropathy and treated in the hind paw with high-dose (15 pg/mouse) BoNT-A devoid of complexing proteins [40]. The reduced astroglial expression supports the hypothesis that BoNT-A may have a modulatory action on glial cells and their role in central nociceptive sensitization [20]. However, BoNT-A effects on glial cells (microglia and astrocytes) should be more carefully explored under pharmacologically relevant moderate doses.
Finally, we assessed possible changes in the peptidergic content in the central afferent terminals by examining the expression of CGRP in the TNC, which was previously found to be increased in the more acute CFA-evoked unilateral monoarthritis of the TMJ [41]. Herein, we did not observe changes in the CGRP expression in the TNC (Figure 6).

Localization of cSNAP-25 in TNC
Formerly, it was assumed that BoNT-A actions resided peripherally, and its antinociceptive activity was linked to a local reduction of the synaptic transmitter/proinflammatory peptide release [18,42]. However, its analgesic effects were shown to be mediated by axonal transport and associated with toxin's enzymatic activity in the CNS [15,17,32]. Previously, we discovered that inhibition of the axonal transport within the peripheral nerve or in the sensory ganglion prevent the antinociceptive effect of peripherally injected BoNT-A, while the toxin injections in the peripheral nerve or in the trigeminal ganglion at doses lower than the ones effective in the periphery produce an antinociceptive effect [15,17,29]. Herein, we confirmed the occurrence of BoNT-A (aboBoNT-A and onaBoNT-A) in the brainstem after i.a. injections aimed at the TMJ (Figure 7). cSNAP-25 appeared as fiber-like processes in the ipsilateral TNC, which is the primary pain-processing area for the orofacial nociceptive input [43]. In accordance with previous studies, we observed no cSNAP-25 in the contralateral TNC, in line with our observation that the toxin is axonally transported primarily via sensory neurons ipsilateral to the BoNT-A injection site and their central projections [32].
A possible transcytosis of the toxin within the central neurons has also been proposed [40,44]. However, this was not corroborated in previous studies employing low to moderate non-systemic onaBoNT-A doses (5-15 U/kg), which suggests that BoNT-A enzymatic activity resides in central afferent terminals of transient receptor potential vanilloid 1-expressing neurons [32]. A lack of immunohistochemical evidence for contralateral occurrence of the toxin's enzymatic activity might suggest an unknown indirect analgesic effect of the toxin on the contralateral side. The exact mechanism of the BoNT-A action on contralateral pain processing remains unknown.

Preclinical Comparison of the Analgesic Efficacy of Different BoNT-A Pharmaceutical Preparations
Currently, there is an intense debate regarding the differences in efficacy of various BoNT-A formulations [45][46][47]. Although both aboBoNT-A and onaBoNT-A are produced from identical Hall A strain synthesizing the A1 toxin subtype; they differ in the composition of complexing auxiliary proteins (onaBoNT-A is composed of the entire 900-kDa complex, while aboBoNT-A has a variable composition of complexing proteins per neurotoxin molecule), as well as the relative potency of the different products [46]. Despite the possibility that these differences also depend on the amount of active toxins in individual preparations [47], they have rarely been studied in both animals and humans. To compare the effect of aboBoNT-A vs. onaBoNT-A, we employed a 2:1 dose conversion ratio according to Scaglione et al. [45]. Following a study from Field et al., who calculated the picogram content of each international unit (U) (5.38 pg of 150-kDa neurotoxin protein for aboBoNT-A, while 1 U of onaBoNT-A contains 9 pg of 150-kDa protein) [47], approx. 400-g rats were herein injected with similar 150-kDa BoNT-A amounts (30.1 pg/animal of aboBoNT-A and 25.2 pg/animal of onaBoNT-A). Despite some possible differences in the total amount of the neurotoxin injected, we found no significant differences in the antinociceptive activity of any investigated outcome between the two BoNT-A preparations (Figures 2-6). This suggests that both products may have similar analgesic effects [48,49].

Limitations
As a limitation of the study, we must consider that this experiment is restricted to the trigeminal region, so differences in the mechanism of action of BoNT-A applied to other extracranial joints (e.g., knee and ankle) and actions at the spinal levels cannot be excluded. Additionally, during the period after BoNT-A treatment, the aboBoNT-A-or onaBoNT-A-treated rats did not exhibit a significantly slower weight gain compared to the saline-treated rat groups (p = 0.070, one-way ANOVA, results not shown). This excludes possible systemic effects or a local muscular BoNT-A action (e.g., mastication and feeding) interfering with the animal weight gain.

Materials and Methods
Animal procedures were approved by the Ethics Committee of the University of Zagreb School of Medicine (permit NoEP 108/2017) and conducted according to the European Communities Council Directive and the International Association for the Study of Pain guidelines [50]. All efforts were made to reduce the number of animals. All data was presented in accordance with the ARRIVE guidelines [51].

Persistent Immunogenic Hypersensitivity (PIH) in the TMJ
Forty male Sprague-Dawley rats (300-400 g, age 6-8 weeks at the start of experiment, University of Zagreb School of Medicine, Zagreb, Croatia) were housed in plastic cages with a maximum of 3 animals per cage. Animals were kept in a room with a controlled constant temperature (22 ± 0.5 • C) and a 12 h:12 h light:dark cycle (lights on from 07:00 to 19:00. Food and water were available ad libitum. Animals were induced to PIH in the TMJ according to previous experiments [8,10]  cluded. Additionally, during the period after BoNT-A treatment, the aboBoNT-A-or onaBoNT-A-treated rats did not exhibit a significantly slower weight gain compared to the saline-treated rat groups (p = 0.070, one-way ANOVA, results not shown). This excludes possible systemic effects or a local muscular BoNT-A action (e.g., mastication and feeding) interfering with the animal weight gain.

Materials and Methods
Animal procedures were approved by the Ethics Committee of the University of Zagreb School of Medicine (permit NoEP 108/2017) and conducted according to the European Communities Council Directive and the International Association for the Study of Pain guidelines [50]. All efforts were made to reduce the number of animals. All data was presented in accordance with the ARRIVE guidelines [51].

Persistent Immunogenic Hypersensitivity (PIH) in the TMJ
Forty male Sprague-Dawley rats (300-400 g, age 6-8 weeks at the start of experiment, University of Zagreb School of Medicine, Zagreb, Croatia) were housed in plastic cages with a maximum of 3 animals per cage. Animals were kept in a room with a controlled constant temperature (22 ± 0.5 °C) and a 12 h :12h light:dark cycle (lights on from 07:00 to 19:00. Food and water were available ad libitum. Animals were induced to PIH in the TMJ according to previous experiments [8,10]  In comparison to PIH-induced animals, the control animals (noninduced to PIH) were similarly treated with mBSA; however, they were not subjected to systemic immunologic adjuvants CFA and IFA. These rats received a s.c. injection of mBSA (500 μg) diluted only in phosphate-buffered saline (PBS, 100 μL) on days 0, 7, and 14. Then, similar to PIH rats, on days 21, 28, and 35, the animals received a unilateral (left) intra-TMJ injection of sterile mBSA (10 μg; i.a.) dissolved in PBS (15 μL). In comparison to PIH-induced animals, the control animals (noninduced to PIH) were similarly treated with mBSA; however, they were not subjected to systemic immunologic adjuvants CFA and IFA. These rats received a s.c. injection of mBSA (500 µg) diluted only in phosphate-buffered saline (PBS, 100 µL) on days 0, 7, and 14. Then, similar to PIH rats, on days 21, 28, and 35, the animals received a unilateral (left) intra-TMJ injection of sterile mBSA (10 µg; i.a.) dissolved in PBS (15 µL).

Study Design
Animals were randomly allocated into four different experimental groups (n = 10). The first group included animals noninduced to PIH that received i.a. injection of saline on day 42. The second group included animals induced to PIH and treated with saline solution into the TMJ on day 42. The third and the fourth groups included animals induced to PIH and treated with either abobotulinumtoxinA or onabotulinumtoxinA into the TMJ on day 42.

Spontaneous and Mechanically Evoked Nocifensive Behaviors
All behavioral evaluations were conducted before (day 13; pre-formalin) and after a low dose of intra-TMJ formalin injection (day 14, post-formalin) in alignment with the time points used in our previous experiment ( Figure 1) [8]. Animals were allowed to accommodate for 10 min inside the testing cages before each evaluation in a quiet environment with the appropriate lighting (between 9 a.m. and 4 p.m.). For the preformalin assessments, animals were placed directly in the testing cage. For the postformalin assessment, animals were briefly anesthetized with isoflurane (5% induction; 2.5% maintenance), injected i.a. with low-dose formalin (0.5%; 15 µL), and immediately returned to the testing cage to recover (recovery time: 1 to 2 min) and to perform the behavioral evaluations in the following order: (1) behavioral nocifensive responses and rat grimace scale evaluated simultaneously and (2) mechanical sensitivity using von Frey filaments. The experimenter who performed the behavioral assessments was blinded to all treatments.

Behavioral Nocifensive Responses
The spontaneous nocifensive responses were assessed for 45 min, as described by previous studies [8,12]. The assessment of the behavioral nocifensive responses was calculated by the sum of seconds the animal spent rubbing or scratching the orofacial region (assessed live by a stopwatch) plus the total number of head flinches (1 flinch = 1 s).

The Rat Grimace Scale (RGS)
A video camera was placed in the upper part of the testing cage in a position that allowed a complete view of each animal. Rats were continuously recorded during the evaluation of their behavioral nocifensive responses (starting after awakening from isoflurane anesthesia) divided across 3 blocks of 5 min with a 4-min interval between each block (0-5 , 9-14 , and 18-23 ). After recording, the videos were analyzed by an experimenter blinded to the treatment. A 0-2 score (0 = not present, 1 = moderate, and 2 = obvious pain) was assigned to each facial parameter (orbital tightening, nose/cheek flattening, ear changes, and whisker changes) observed in each 5-min block, as previously described [52]. A mean of the scores in all blocks was considered the total RGS score.

Mechanical Sensitivity to von Frey Filaments
Mechanical allodynia was assessed immediately after the completion of the assessment of spontaneous behavioral nociceptive responses by using von Frey monofilaments (Stoeling Co., Wood Dale, IL, USA). The assessment of mechanical allodynia lasted for 5-10 min. The test was performed as previously described [29]. The filaments (flat contact area and weights in grams: 0.16, 0.4, 0.6, 1, 1.4, 2, 4, 6, 8, and 10) were applied bilaterally over the TMJ area, starting with the contralateral nontreated TMJ (right TMJ). In each session, each filament was applied three times (starting with 0.16-g filament and continuing in ascending order) until a repeated positive response (escape and/or defensive movement) after the stimulation of the evaluated area was elicited. The maximum weight (10 g) was assigned when no response was observed.

Immunohistochemistry
On day 57, immediately after behavioral assessments, all animals were deeply anesthetized (ketamine 70 mg/kg and xylazine 7 mg/kg, intraperitoneally (i.p.)) and transcardially perfused with saline followed by buffered 4% paraformaldehyde (PFA) in PBS (pH 7.4). The brain was dissected and placed in the PFA solution overnight (18-20 h) at +4 • C. Then, the samples were transferred to 30% sucrose dissolved in PBS for 48h at +4 • C. Then, all samples were stored at −80 • C prior to further use. The caudal part of the medulla oblongata containing the trigeminal nucleus caudalis (TNC; from −5.28 mm to −6.72 interaural, based on Paxinos and Watson [53]) was cut into 35-µm coronal cross-sections using a cryostat. The position of the sections relative to the bregma/interaural line was determined based on visible landmarks such as the obex, position of the central canal, and shape of the white and gray matter, according to the rat brain atlas [53]. For each immunohistochemical analysis, 5 sections per animal were randomly chosen.

Localization of cSNAP-25 in the Brainstem
Sections corresponding to the TNC region of the brainstem were randomly selected, washed in PBS with 0.25% Triton X-100 (PBST), and exposed to 50 µL of 3% peroxide diluted in 200-µL PBS for 1h. Sections were washed again and blocked with 10% normal goat serum (NGS) in PBST. A well-characterized rabbit polyclonal antibody that specifically cleaves the BoNT-A-truncated SNAP-25 (nonaffinity-purified rabbit antiserum (anti-SNAP-25 197 , National Institute for Biological Standards and Control, Potters Bar, UK, a generous gift from Dr. Thea Sesardic) was diluted in 1% NGS (1:4000), and samples were incubated overnight at room temperature. The next day, the sections were washed, and 200 µL of Alexa Fluor 555 secondary antibody (1:400, Molecular Probes, Invitrogen, Carlsbad, CA, USA) was used to enhance the cSNAP-25 signal. Samples were then washed 3 times before mounting into the glass slide and coverslipped with an antifading agent. All samples were visualized with an epifluorescent microscope (Olympus BX-51, Tokyo, Japan). Fiber-shaped staining was searched, and pictures were taken using a digital camera (DP-70, Olympus, Tokyo, Japan).

Quantification of Neuronal and Astrocyte Activation in the Brainstem
The brainstem sections of each animal containing the TNC were randomly selected for immunohistochemical analyses. The sections were placed in free-floating plate wells in PBS with PBST and washed and blocked with 10% NGS in PBST. Sections of TNC were incubated overnight at room temperature in 1% NGS with either anti-c-Fos Immunostained sections were visualized with an Olympus BX-51 epifluorescent microscope coupled to a DP-70 digital camera (Olympus, Tokyo, Japan), and brain regions were identified using the rat stereotaxic atlas [53]. All images were processed and quantified by using cellSens Dimension Software (Olympus, Tokyo, Japan). The c-Fos-positive neurons were automatically counted in the ipsilateral and contralateral side of the dorsal horns of each section (TNC). For the acquisition of GFAP, microphotographs of each coronal section containing the TNC was taken ipsilaterally at a 10× objective magnification and 0.5× camera adapter, while for CGRP quantification, we employed 4× objective magnification and a 0.5× camera adapter. A constant exposure was used to acquire individual microphotographs for each subsequent quantification, as well as the manual thresholds used for calculation of the object number and immunoreactivity areas. The immunohistochemical quantification of the area of immunoreactivity, as well as the number of objects within the area of interest, were based on manual pixel thresholding of grey images obtained from separated RGB channels: red or green based on the fluorophore used (pixel threshold range 115-256 for GFAP and c-Fos images taken at 10× objective magnification; range 62-256 for CGRP microphotographs taken at 4× objective magnification).

Statistical Analysis
Quantitative data were presented as the mean ± standard error of the mean (SEM). Data from repeated behavioral measurements were analyzed using the two-way repeated measures analysis of variance (RM ANOVA), followed by Bonferroni's post hoc test to compare the intertreatment effects. Single time observations were analyzed by one-way ANOVA, followed by Tukey's post hoc test. The data representing mechanical sensitivity, assessed by Von Frey filaments, were log-transformed prior to statistical analysis, according to the fact that mechanical sensation is perceived on a logarithmic scale (Weber's law) [54].
All statistical analyses were performed with GraphPad Prism version 5 (GraphPad Software, Inc., San Diego, CA, USA). A p-value < 0.05 was considered significant.

Conclusions
Based on the present data and previous results [8], the analgesic activity of BoNT-A in rats exposed to a PIH in the TMJ seems to be associated with peripheral and central sites of BoNT-A action. The present results suggest that BoNT-A action is associated with a reduction of spontaneous and evoked nociceptive pain measures suggestive of central sensitization (reduction of pain grimacing in the later assessment phase similar to the BoNT-A action on phase II of the formalin test and reduction of bilateral mechanical allodynia). In addition, we found that the antinociceptive effect of BoNT-A is accompanied by reduction of neuronal and astrocytic activation in the TNC, both effects associated with central pain processing. The mentioned actions did not significantly differ with respect to different pharmacological preparations of the toxin (aboBoNT-A vs. onaBoNT-A). In addition, the antinociceptive activity of aboBoNT-A and onaBoNT-A was accompanied by the occurrence of central cSNAP-25, suggestive of the toxin's direct central action. However, the relative importance of each site of action (peripheral vs. central) for this effect remains to be further investigated in the present pain model.  Institutional Review Board Statement: Animal procedures were approved by the Ethics Committee of the University of Zagreb School of Medicine and Croatian Ministry of Agriculture (permit No EP 108/2017) and conducted according to the European Communities Council Directive and the International Association for the Study of Pain guidelines. All efforts were made to reduce the number of animals. All data were presented in accordance with the ARRIVE guidelines.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.


The Roles of Histone Post-Translational Modifications in the Formation and Function of a Mitotic Chromosome

During mitosis, many cellular structures are organized to segregate the replicated genome to the daughter cells. Chromatin is condensed to shape a mitotic chromosome. A multiprotein complex known as kinetochore is organized on a specific region of each chromosome, the centromere, which is defined by the presence of a histone H3 variant called CENP-A. The cytoskeleton is re-arranged to give rise to the mitotic spindle that binds to kinetochores and leads to the movement of chromosomes. How chromatin regulates different activities during mitosis is not well known. The role of histone post-translational modifications (HPTMs) in mitosis has been recently revealed. Specific HPTMs participate in local compaction during chromosome condensation. On the other hand, HPTMs are involved in CENP-A incorporation in the centromere region, an essential activity to maintain centromere identity. HPTMs also participate in the formation of regulatory protein complexes, such as the chromosomal passenger complex (CPC) and the spindle assembly checkpoint (SAC). Finally, we discuss how HPTMs can be modified by environmental factors and the possible consequences on chromosome segregation and genome stability.

Introduction
In eukaryotic cells, the DNA is bound to histone and non-histone proteins in a complex known as chromatin. In total, 147 base pairs of DNA are wrapped on a histone octamer formed by two copies of each of the core histones (H2A, H2B, H3, and H4). This unit is known as a nucleosome. Between nucleosomes, there are stretches of DNA bound to the linker histone H1 [1]. Core histones are constituted by a largely alpha-helical C-terminal domain, which is involved in the formation of the octamer and the DNA binding. The N-terminal region of core histones is organized in an unstructured tail. These tails and the C-terminal of H2A are the sites where most post-translational modifications are found [1,2]. Histones, like other proteins, can be modified in specific residues with the addition of different molecules; they can be acetylated, methylated, phosphorylated, and ubiquitinated, among other modifications. These modifications can change their affinity for DNA or can be "read" by other proteins and recruit specific factors to the nucleosome. Lysine acetylation was the first HPTM described. It opens condensed chromatin by promoting a charge neutralization in histones. Acetylated histones form a more relaxed chromatin which facilitates the recruitment of transcription machinery. Moreover, lysine acetylation is recognized by the bromodomain of several proteins that regulate gene expression. Lysine acetylation is also involved in DNA repair. This mark is deposited by the histone acetyltransferases (HATs) and removed by the histone deacetylases (HDACs) [3,4].
Histone lysines and arginines can be methylated by enzymes that use S-adenosyl-L-methionine as a methyl donor. Lysines can be mono-, di-, or trimethylated, while arginines can be mono-or dimethylated. The role of lysine methylation in transcriptional regulation is well known. Lysine methylation is "read" by chromodomain-containing proteins, which can promote or repress transcription [5]. Lysine methylation is removed by lysine-specific demethylases (LSDs) or members of the Jumonji family of demethylases. On the other hand, the arginine methylation of histones is also associated with the regulation of gene expression [2]. The effect of histone methylation can differ depending on the position of the methylated residue and its level (mono-, di-, or trimethylated) [6,7]. Histone phosphorylation is an important mark for the DNA damage response (DDR). Histone phosphorylation is also associated with transcription regulation [2,8]. Finally, histone ubiquitination is another mark that has been associated with transcription regulation and DDR [9]. It has been proposed that histone modifications in the same histone tail or nearby tails from different histones can constitute a "code" that is interpreted to perform different functions in chromatin. Although the regulation of gene expression and DDR using histone HPTMs has been extensively studied, the role of histone PTMs in chromosome segregation has recently been revealed.
In addition to transcriptional regulation and DNA repair, HPTMs can play other structural and functional roles along the cell cycle. Particularly in mitosis, HPTMs are involved into the chromatin condensation, forming visible chromosomes under the microscope. Chromosome condensation is necessary to facilitate the transport and separation of genetic material to daughter cells. In this process, chromatin reduces its length by up to four orders of magnitude, significantly modifying its structure and physical properties [10]. Each chromosome harbors a region known as centromere, whose identity is defined by the presence of the distinctive H3 histone variant CENP-A, as well as several accompanying HPTMs [11]. The centromere is important because a multiprotein complex known as kinetochore is assembled onto that region, and it binds microtubules to align the chromosomes [12]. In addition, specific HPTMs also regulate the spindle assembly checkpoint (SAC) and the activity of the chromosomal passenger complex (CPC). The SAC checks the union between microtubules and kinetochores and stops mitosis until all chromosomes are attached to microtubules from the mitotic spindle. On the other hand, the CPC corrects kinetochore-microtubule attachment errors and participates in the contractile ring formation during cytokinesis [13,14]. Chromatin and specific HPTMs are involved in several of these processes during mitosis.
Remarkably, HPTMs are not only modified by cellular intrinsic factors but also by extrinsic environmental factors. For example, environmental pollutants can also modify HPTMs and disturb some of the mechanisms they are involved in [15]. Chromosome segregation is one potential mechanism that can be disturbed by environmental pollutants since histone PTMs are involved in its regulation.
In this review, we focus on the role of histone PTMs in mitosis, from chromosome condensation to the regulation and activity of the centromere and kinetochore. We also discuss how environmental factors could modify histone PTMs and disturb chromosome segregation.

Chromosome Condensation
During mitosis, chromatin is condensed to form discrete units known as mitotic chromosomes. Condensation facilitates the segregation of the exceptionally long DNA molecules, which form chromosomes. Many factors have been associated with chromosome condensation, but the specific mechanism whereby chromatin is condensed in a mitotic chromosome is not well understood. In an in vitro model of chromosome condensation, six factors were shown to be essential: core histones, nucleoplasmin, Nap1, FACT, topoisomerase II (topo II), and condensin I [16]. Although these purified factors could form mitotic chromosomes, it is unknown if condensation is achieved in a normal way because compaction was not quantified in this assay.
It is known that nucleosomes are essential for chromosome condensation, although the role of histone modifications in this mechanism is in debate. The phosphorylation of histone H3 at serine 10 (H3S10ph) using the Aurora B kinase is noticeable since prophase, and it has been associated with chromosome condensation [17,18]. However, several reports have challenged the role of H3S10ph in chromosome condensation. The mutation of serines 10 and 28 of H3 does not alter chromosome condensation in yeast [19]. Furthermore, chromosome condensation is not significantly modified in Drosophila and human cells when Aurora B is inhibited [20,21]. Finally, incubation with a hypotonic solution can promote H3S10 dephosphorylation in HeLa cells without an observable difference in chromosome structure in comparison with cells that retain the H3S10ph mark [22]. However, some data support the role for H3S10ph in chromosome condensation. There is a cascade of histone modifications during mitosis that promotes chromatin compaction via the interaction between adjacent nucleosomes in yeast. H3S10ph recruits Hst2p (a histone deacetylase), which in turn deacetylases the lysine 16 of H4 (H4K16ac). The deacetylation of H4K16 allows the contact between the N terminus of H4 and the dimer interface of H2A-H2B in the adjacent nucleosome, promoting chromatin compaction ( Figure 1) [23]. Moreover, Barral et al. have demonstrated the role of H3S10ph in chromatin compaction during mitosis but not in the axial shortening of chromosomes in yeast [24]. It is important to note that these studies have been performed in yeast with relatively small chromosomes. A study with mammalian chromosomes showed that the highest level of compaction occurred during anaphase and that it depends on axial shortening mediated by Aurora B activity. However, whether such activity depended on H3S10ph or another substrate of Aurora B was not established [25].  Figure 1) [30]. On the other hand, in an in vitro model, the N terminus of H2B was show to be essential to achieve chromosome condensation, and the N terminus of H2A and H was also important to condensation, but to a lesser extent [31]. Changes in the methylation of the H3K9 residue during mitosis have also been found In mammalian cells, H3K9 methylation levels decrease during mitosis (mono-, di-, an trimethylation), especially for H3K9Me3 [32][33][34]. However, some methylation levels a H3K9 are necessary for segregation because their inhibition causes defects in chromosom segregation [34]. By using FRET biosensors, it was demonstrated that H3S10 phosphory lation causes H3K9Me3 to decrease. Furthermore, this study showed that the H3K9Me decrease is associated with the dissolution of heterochromatin structures, which they pro pose is necessary for proper global chromosome compaction in mitosis [32]. Moreove the decrease in H3K9Me3 coincides with an increase in global histone H1 phosphoryla tion. However, the role of this change in chromosome formation and segregation is un clear [33]. H3 is phosphorylated in th serine 10 along all the chromosomes. This modification recruits Hst2p, which deacetylates H4K1 by inducing local nucleosome joining and local chromatin compaction. H3T118 also participates i chromosome condensation, and its distribution is preferentially in the pericentromeric chromatin but its function is still unknown.

Histone Modifications, CENP-A Incorporation, and Kinetochore Function
The centromeric chromatin is recognized to contain a histone modification patter different from those classically related to active or repressive chromatin. Karpen and Su livan coined the term "centrochromatin" to refer to this specialized type of chromati Condensins participate in the axial shortening of chromosomes while histone modifications allow the local compaction. H3 is phosphorylated in the serine 10 along all the chromosomes. This modification recruits Hst2p, which deacetylates H4K16 by inducing local nucleosome joining and local chromatin compaction. H3T118 also participates in chromosome condensation, and its distribution is preferentially in the pericentromeric chromatin, but its function is still unknown.
Although several reports have raised controversy about the specific role of H3S10ph in chromosome condensation, it is important to note that most of these studies are only qualitative, and chromosome condensation is visually evaluated as the formation of a typical chromosome without quantifying the level of condensation. Recently, fluorescencebased techniques have been used to measure chromosome condensation by quantifying the distance between two fluorescent marks in a chromosome (axial shortening) or the measurement of the fluorescence level of a region where lower fluorescence is associated with a more compacted chromatin due to a quenching effect (chromatin compaction) [24,26]. Using these techniques, it has been demonstrated that chromosomes are compacted by two independent pathways, first by local compaction, which is due to changes in histone modifications, and then by axial shortening, which depends on the action of Condensins.
The function of Aurora B kinase in the formation of mitotic chromosomes has been demonstrated in different studies. In chicken DT40 cells, it was demonstrated that a factor (RCA regulator of chromosome architecture) maintains chromosome structure independently of condensins [27]. This factor is inhibited by Repo-Man-PP1γ, a complex whose phosphatase activity eliminates H3 phosphorylation by Aurora B [28]. There is a negative feedback between Repo-Man-PP1γ, and Aurora B. Repo-Man-PP1γ removes the H3T3ph tag required for CPC recruitment, whereas Aurora B phosphorylates Repo-Man, preventing PP1γ recruitment to the chromosome [28,29]. Although the role of Aurora B activity in chromosome condensation is clear, it has not been shown to depend exclusively on its kinase activity on the H3S10 residue. Repo-Man-PP1γ was shown to participate mainly in the dephosphorylation of H3T3ph and H3T11ph [28].
The aurora A-dependent phosphorylation of threonine 118 on H3 (H3T118ph), which occurs at the histone DNA interface, is present in mitotic chromosomes from prophase until metaphase and is also associated with chromosome condensation in metazoans. The expression of the phosphomimetic mutant H3 T118I or Aurora A overexpression cause the shortening and widening of chromosome arms. Moreover, the mutant H3 T118I diminished the Condensin I association with chromatin through an unknown mechanism ( Figure 1) [30]. On the other hand, in an in vitro model, the N terminus of H2B was shown to be essential to achieve chromosome condensation, and the N terminus of H2A and H4 was also important to condensation, but to a lesser extent [31].
Changes in the methylation of the H3K9 residue during mitosis have also been found. In mammalian cells, H3K9 methylation levels decrease during mitosis (mono-, di-, and trimethylation), especially for H3K9Me3 [32][33][34]. However, some methylation levels at H3K9 are necessary for segregation because their inhibition causes defects in chromosome segregation [34]. By using FRET biosensors, it was demonstrated that H3S10 phosphorylation causes H3K9Me3 to decrease. Furthermore, this study showed that the H3K9Me3 decrease is associated with the dissolution of heterochromatin structures, which they propose is necessary for proper global chromosome compaction in mitosis [32]. Moreover, the decrease in H3K9Me3 coincides with an increase in global histone H1 phosphorylation. However, the role of this change in chromosome formation and segregation is unclear [33].

Histone Modifications, CENP-A Incorporation, and Kinetochore Function
The centromeric chromatin is recognized to contain a histone modification pattern different from those classically related to active or repressive chromatin. Karpen and Sullivan coined the term "centrochromatin" to refer to this specialized type of chromatin [35,36]. However, the real diversity of HPTMs present in this region and their specific roles are only starting to be uncovered. Recent data show that the centromeric chromatin context is a key determinant of proper mitotic progression [37], since it bears HPTMs that promote both the correct function of the inner centromere, through the regulation of the chromosomal passenger complex and the assembly of the kinetochore through the control of CENP-A centromeric incorporation.

HPTMs and the Chromosomal Passenger Complex
The CPC is composed of Survivin, Borealin, INCENP, and the kinase Aurora B, and it is involved in the control of the mitotic chromosome structure, spindle assembly checkpoint regulation, microtubule-kinetochore union, the formation of the mitotic spindle, and cytokinesis [38,39]. The mechanism by which the CPC is located to chromosome arms to induce mitotic chromosome structure has been elusive until recently when the asymmetrical dimethylation of arginine 2 was detected on H3 in human cells [40]. H3R2me2a, brought about by the methyltransferase PRMT6, is abundant from the S phase until the M/G1 transition. This HPTM was shown to participate in the recruitment of the CPC to chromosome arms during the late G2/early prophase. The relation between H3R2me2a, the CPC, H3S10ph, and mitotic chromosome condensation was determined by tomographic microscopy. In the same study, the authors reported that while the cohesion of both mitotic chromosome arms and centromeres rely upon H3R2me2a, the former depends on CPC recruitment and H3S10ph, while the latter is mediated by the centromeric localization of Shugoshins 1 and 2 [40].
In terms of SAC regulation, the CPC is responsible for the destabilization of incorrect kinetochore-microtubule attachments [41]. Unattached kinetochores result in the activation of the SAC, with a consequential mitotic arrest. CPC function depends on its correct localization in metaphase, which in turn relies upon two mitosis-specific histone marks. During metaphase, this complex is recruited to the inner centromere by the phosphorylation of histone H3 at threonine 3 and the phosphorylation of histone H2A at threonine 120 [42]. These marks have a different distribution throughout mitotic chromosomes: H3T3ph is observed along the entire arms of the chromosome, proximal to the site where both sister chromatids contact each other, while H2AT120ph is found along the axis that connects the kinetochores of two sister chromatids [42]. Interestingly, it has been shown that the CPC is recruited to the intersection of both histone phosphorylations, i.e., the inner centromere ( Figure 2). The phosphorylation of H3T3 by the kinase Haspin [43] is recognized and bound by a pocket in the BIR domain of Survivin [44], while H2AT120ph recruits the CPC through Shugoshin1. This protein directly binds to the phosphorylated residue and functions as an adaptor by contacting Borealin, thus tethering the CPC to the site of H2AT120ph in an indirect manner [45]. Furthermore, inhibiting the histone phosphorylations that recruit the CPC to the inner centromere during metaphase by depleting the kinases responsible for them or preventing their recognition with the use of specific antibody microinjection causes the delocalization of the CPC and consequently has detrimental effects on mitotic progression [42,43].
chromosomal passenger complex and the assembly of the kinetochore through the control of CENP-A centromeric incorporation.

HPTMs and the Chromosomal Passenger Complex
The CPC is composed of Survivin, Borealin, INCENP, and the kinase Aurora B, and it is involved in the control of the mitotic chromosome structure, spindle assembly checkpoint regulation, microtubule-kinetochore union, the formation of the mitotic spindle, and cytokinesis [38,39]. The mechanism by which the CPC is located to chromosome arms to induce mitotic chromosome structure has been elusive until recently when the asymmetrical dimethylation of arginine 2 was detected on H3 in human cells [40]. H3R2me2a, brought about by the methyltransferase PRMT6, is abundant from the S phase until the M/G1 transition. This HPTM was shown to participate in the recruitment of the CPC to chromosome arms during the late G2/early prophase. The relation between H3R2me2a, the CPC, H3S10ph, and mitotic chromosome condensation was determined by tomographic microscopy. In the same study, the authors reported that while the cohesion of both mitotic chromosome arms and centromeres rely upon H3R2me2a, the former depends on CPC recruitment and H3S10ph, while the latter is mediated by the centromeric localization of Shugoshins 1 and 2 [40].
In terms of SAC regulation, the CPC is responsible for the destabilization of incorrect kinetochore-microtubule attachments [41]. Unattached kinetochores result in the activation of the SAC, with a consequential mitotic arrest. CPC function depends on its correct localization in metaphase, which in turn relies upon two mitosis-specific histone marks. During metaphase, this complex is recruited to the inner centromere by the phosphorylation of histone H3 at threonine 3 and the phosphorylation of histone H2A at threonine 120 [42]. These marks have a different distribution throughout mitotic chromosomes: H3T3ph is observed along the entire arms of the chromosome, proximal to the site where both sister chromatids contact each other, while H2AT120ph is found along the axis that connects the kinetochores of two sister chromatids [42]. Interestingly, it has been shown that the CPC is recruited to the intersection of both histone phosphorylations, i.e., the inner centromere ( Figure 2). The phosphorylation of H3T3 by the kinase Haspin [43] is recognized and bound by a pocket in the BIR domain of Survivin [44], while H2AT120ph recruits the CPC through Shugoshin1. This protein directly binds to the phosphorylated residue and functions as an adaptor by contacting Borealin, thus tethering the CPC to the site of H2AT120ph in an indirect manner [45]. Furthermore, inhibiting the histone phosphorylations that recruit the CPC to the inner centromere during metaphase by depleting the kinases responsible for them or preventing their recognition with the use of specific antibody microinjection causes the delocalization of the CPC and consequently has detrimental effects on mitotic progression [42,43]. Figure 2. HPTM-dependent CPC recruitment. H3T3 and H2AT120 phosphorylations, by Haspin and BUB1, respectively, are two key HPTMs necessary for the recruitment of the CPC to centromeres. The coincidence of these marks on mitotic chromosomes specifies the site of CPC localization. Survivin binds to H3T3ph, conveying the other components of the complex, while Shugoshin binds to H2AT120, acting as an adaptor between this mark and Borealin. A phosphorylationdependent positive feedback loop occurs between Aurora B and Haspin kinases, reinforcing the CPC recruitment pathway.

HPTMs and CENP-A Centromeric Incorporation
On the other hand, CENP-A, whose recruitment is also highly dependent on HPTMs, is the most critical protein of kinetochore assembly. It is a histone H3 variant and the distinctive epigenetic element of active centromeres [11]. CENP-A is the interface between the centromere and the constitutive centromere-associated network (CCAN), a large protein complex that directly tethers to microtubules during chromosome segregation [46]. Accordingly, CENP-A recruitment is tightly regulated, and the same amount is incorporated into the centromere in every cell cycle in normal conditions [11].
CENP-A cellular production occurs during the late synthesis/G2 phase of the cell cycle. However, its centromeric incorporation is prevented until the next G1 by inhibitory phosphorylations on HJURP, Mis18BP1, and CENP-A itself (see below), mediated by the CDK1 and CDK2 kinases. Instead, the nucleosome gaps in the centromeric chromatin resulting from the distribution of CENP-A among the old and newly synthesized DNA during the S phase are filled with histone H3.1-and histone H3.3-containing nucleosomes. In the next G1 phase, histone H3.3-containing nucleosomes are exchanged for nucleosomes containing newly synthesized CENP-A [47][48][49]. Notably, the machinery responsible for CENP-A incorporation ultimately relies on the coordination of several HPTMs on different histones within specific time frames. Such modifications have been shown to occur on histones H2B, H3, and H4, as well as CENP-A itself.
Histone H3 from nucleosomes adjacent to CENP-A nucleosomes was recently purified and analyzed by mass spectrometry. This allowed the identification of a complex mixture of modifications, including the acetylation of residues K14, K18, and K23, and the mono-, di-, and trimethylation of residues K4, K9, K27, and K36 [35]. Of note, the presence of CENP-A in centromeric nucleosomes is mutually exclusive with that of canonical H3 [50][51][52][53]. However, CENP-A-containing nucleosomes are interspersed with those containing H3 [36]. Therefore, CENP-A-associated chromatin contains histone H3 from nucleosomes neighboring those that do contain CENP-A. While centromeric H3 is enriched in methylation, its acetylation was found to be rather scarce [35]. In agreement with this, H3K9 trimethylation impedes centromeric nucleosome turnover while H3K9 acetylation permits it, and this process occurs exclusively during late mitosis/early G1 [54]. Therefore, the observation that the centromere bears low acetylation levels seems to be a result of the temporal regulation to which this HPTM is subjected. Accordingly, centromere acetylation takes place only during a short time frame at such a point of the cell cycle, and this results in low levels of centromere acetylation detection in asynchronous cells [54].
Further support for the importance of centromeric chromatin HPTMs comes from work in the Larionov, Earnshaw, and Masumoto laboratories. Using human artificial chromosomes (HACs), they developed a strategy to evaluate the function of chromatin modifiers localized directly on the centromere [55]. Their studies revealed that the excess of transcriptional activity formed by transcriptional activators and the excess of heterochromatin formed by transcriptional silencers could affect kinetochore assembly by the improper incorporation of H3K4me2 (associated with active transcription) and H3K9me3 (associated with transcriptional repression), respectively [55]. The overexpression of LSD1, an H3K4 histone demethylase localized directly to the centromere, reduces H3K4me2 presence in this region. Furthermore, the modification of H3K4me2 levels in the centromeric regions impedes the incorporation of CENP-H and CENP-C, HJURP, and CENP-A, therefore hindering correct kinetochore function [56,57].
On the other hand, it has been shown that CENP-A assembly can be achieved at ectopic alphoid DNA regions in several human cell lines. However, these CENP-A arrays are commonly lost, and their loss is accompanied by an increase in the trimethylation of histone H3 at lysine 9 [54]. Interestingly, the abundance of both H3K9me3 and H3K9ac can vary in different cell lines, and the balance between these two marks is associated with the propensity to retain ectopic CENP-A arrays. For instance, the cell line HT1080 has a higher tendency to retain such arrays than HeLa, TIG7, hTERT-BJ1, and U2OS. This is associated with the H3K9me3/H3K9ac ratio, which is lower in HT1080 cells [54]. Furthermore, the depletion of Suv39h1, an H3K9 methyltransferase, increases CENP-A incorporation to the ectopic array, while ectopically targeting acetyltransferases to an alphoid DNA array promotes de novo kinetochore formation in HeLa cells. Thus, the presence of either the methylation or acetylation of H3 at the centromere is determinant in the maintenance of a functional centromere [54].
Moreover, centromere acetylation is the result of an interesting cascade of events involving the centromeric localization of the Mis18 complex ( Figure 3). Some lines of evidence strongly suggest that the central role of this complex in CENP-A incorporation is the priming of centrochromatin [11]. In human cells, the Mis18 complex, which is composed of hMis18α, hMis18β, and hMis18BP1, is specifically accumulated at the telophase-G1 centromere [58] and promotes the recruitment of the KAT7 histone acetyltransferase, which, in turn, acetylates histone H3 on lysine 14 [59]. The depletion of Mis18 complex subunits has been rescued by the treatment of cells with the histone deacetylase inhibitor trichostatin A [60] and by the specific targeting of chimeric histone acetyltransferases to an exogenous centromere [54]. The acetylation of centrochromatin can elicit changes in the chromatin state in at least three different aspects: it promotes chromatin relaxation, which facilitates the eviction of histone H3-containing nucleosomes and the incorporation of CENP-Acontaining nucleosomes; it recruits the remodeling and spacing factor 1 (RSF1), a protein required for the stabilization of CENP-A at the centromeric array [59]; and it also permits centromeric transcription which gives rise to non-coding RNAs derived from alpha satellite, which have been shown to participate in HJURP-mediated CENP-A incorporation to the centromere (Figure 3) [61]. Centromere H3 acetylation is not the only HPTM associated with centromeric transcription around the time of CENP-A assembly. A transient pulse of centromeric H2B monoubiquitination is brought about by the ubiquitin ligase RNF20 in mitosis [62]. This histone modification stimulates the RNA pol-II-mediated transcription of the centromere. Previous work has shown that H2BK120ub1 promotes H3K4 di-and trimethylation through the hSET1 complex [63], and H3K79 methylation mediated by hDOT1L [64]. H3K4me2/3 is related to the transcription start site of active genes, while H3K79me has been found in the body of active genes [65], providing a mechanistic explanation for the observed increase in centromeric transcription. These changes, elicited by H2BK120ub1, decrease histone H3 stability at the centromere, facilitating CENP-A incorporation. Furthermore, inhibiting H2BK120ub1 by the depletion of RNF20 with the use of RNAi is associated with an increase in silencing histone modifications and a decrease in centromeric transcription and histone turnover, thus affecting CENP-A assembly. Naturally, this phenotype has detrimental consequences on chromosome segregation [62].
Another HPTM important for the centromere structure and kinetochore assembly is H4K20me1 [66]. PR-Set7 is the enzyme responsible for H4K20 methylation, and its regulation is cell-cycle-dependent [67]. During the interphase, H4K20me1 is preferentially associated with the 5 end of actively transcribed genes [68]. However, during late mitosis, H4K20me1 is located on the CENP-A nucleosomes, and this modification occurs after CENP-A incorporation. Recent studies have demonstrated that an increase in PHF8 (an H4K20me1 histone demethylase) inhibits CENP-A-H4K20me1 mature nucleosome formation, reducing CENP-H and CENP-T localization to centromeres and promoting kinetochore assembly defects [66].
On the other hand, the RbAp46/RbAp48 complex recognizes the CENP-A/H4 prenucleosomal dimer and recruits the histone acetyl transferase Hat1, which catalyzes H4K5 and H4K12 acetylation [69,70]. Using RbAp48 depletion, this process was shown to be required for CENP-A deposition. Based on a mutational analysis, the authors of this study also proposed that the failure to neutralize the H4K5-and H4K12-positive charges explains the CENP-A incorporation errors observed in RbAp48-depleted cells. Specifically, they suggested that the acetylation-free H4 tail could interfere with the Mis18 complex-HJURP interaction, therefore hindering proper CENP-A nucleosome centromeric deposition [70]. Another HPTM important for the centromere structure and kinetochore assembly is H4K20me1 [66]. PR-Set7 is the enzyme responsible for H4K20 methylation, and its regulation is cell-cycle-dependent [67]. During the interphase, H4K20me1 is preferentially associated with the 5′ end of actively transcribed genes [68]. However, during late mitosis, H4K20me1 is located on the CENP-A nucleosomes, and this modification occurs after CENP-A incorporation. Recent studies have demonstrated that an increase in PHF8 (an H4K20me1 histone demethylase) inhibits CENP-A-H4K20me1 mature nucleosome formation, reducing CENP-H and CENP-T localization to centromeres and promoting kinetochore assembly defects [66]. Post-translational changes on CENP-A itself are also required for its correct incorporation to the centromere. However, a comprehensive model of the roles played by each of the PTMs discovered on this histone variant is difficult to construct due to conflicting experimental evidence.
Although the sequence of the CENP-A N terminus shows poor conservation, the presence of potentially phosphorylatable serines is a conserved feature of the CENP-A N terminus from this domain among diverse eukaryotic species [71]. For example, CENP-A is phosphorylated on serine 7 by Aurora kinases during mitosis. This modified CENP-A has been shown to localize to the contractile ring and participate in the completion of cytokinesis [72]. Interestingly, CENP-A depletion can be rescued by the expression of a mutant CENP-A in which the amino tail was replaced by the canonical H3 amino tail, which also contains two serines, in positions 10 and 28. These residues are known to be phosphorylated during mitosis [73]. CENP-A amino tail phosphorylation was proposed to be necessary for kinetochore function by recruiting 14-3-3 proteins, which would, in turn, bind CENP-C to promote its correct localization [71]. A more recent study demonstrated that the depletion of endogenous CENP-A could be rescued by a mutant CENP-A in which serine 7 was replaced by a non-phosphorylatable alanine, with no significant appearance of lagging chromosomes, misaligned chromosomes, micronuclei, or altered CENP-C localization. Nonetheless, replacing four serines (including S7, S16, and S18) with non-phosphorylatable alanines or phosphomimetic glutamines in the CENP-A aminoterminal tail only partially rescued the CENP-A depletion in terms of CENP-C recruitment and cell proliferation [69]. The results of these manipulations are similar to the outcome of removing the entire CENP-A N terminus [69,71]. Therefore, CENP-A amino tail phosphorylation seems to be necessary for its centromere incorporation and for proper mitotic completion, but the specific contribution of S7 phosphorylation is still controversial.
Furthermore, the exogenous expression of a mutant CENP-A, in which serines 16 and 18 were replaced by non-phosphorylatable alanines, caused the appearance of lagging and misaligned chromosomes in human dividing cells [74]. Conversely, CENP-A bearing the individual replacement of S16 or S18 for alanine had no significant effects on chromosome segregation [48,74]. Further evidence suggested that S16ph and S18ph promote centromeric chromatin higher-order organization. In another study, S18ph, via the CDK2/cyclin E complex, was shown to inhibit premature CENP-A centromeric deposition. This modification prevents CENP-A's association with HJURP, restricting CENP-A centromeric incorporation to late mitosis/early G1. Moreover, the S18D phosphomimetic mutation of CENP-A hinders its deposition and promotes chromosome instability and tumorigenesis [48]. Together, these data suggest that a single PTM can have different functions along the CENP-A cycle.
Serine 18 is not the only "stop sign" that prevents premature CENP-A centromeric incorporation. It has been shown that serine 68 is phosphorylated by CDK1 during mitosis [49]. Some studies suggest that this residue is necessary for CENP-A recognition by HJURP [49,75]. According to one model, S68 phosphorylation disrupts this interaction during mitosis, and S68 is then removed by PP1γ at the end of this phase, licensing CENP-A's association with HJURP and, consequently, promoting its opportune deposition at centromeres [49]. However, conflicting evidence states that S68 phosphorylation is neither sufficient nor necessary for CENP-A loading, centromere function, or cell viability [76]. Interestingly, S68 is evolutionarily conserved in most eukaryotes [49]. Replacing the corresponding amino acid in mice (i.e., S62) for glutamine (which precludes an interaction between CENP-A and a cleft in HJURP) or glutamate (which partially mimics phosphorylated serine 68) has been shown to impair CENP-A deposition and cell viability, and to cause embryonic lethality [75,77].
Moreover, the CENP-A-HJURP interaction is mediated by distinct binding inter-faces in different species. For instance, human HJURP seems to rely heavily on CENP-A's CENP-A targeting domain (CATD) to recognize this histone while, in chickens, A59 (which corresponds to human S68 but is non-phosphorylatable) is critical for CENP-A-HJURP interaction [78].
Therefore, although it has been established that the essentiality of S68 differs between species, whether its PTMs are required for long-term centromere function is still a matter of debate [49,76,79].
CENP-A is also ubiquitinated on lysine 124 via the CUL4 E3 ubiquitin ligase in complex with COPS8 and RBX1 during mitosis/G1 [80]. This HPTM was shown to be necessary for the correct incorporation of CENP-A, since it promotes its interaction with its chaperone HJURP. Furthermore, substituting lysine 124 by arginine hinders CENP-A centromeric recruitment [80]. As in the case of CENP-A-S68ph, the contribution of K124 ubiquitination to long-term centromere function is controversial [76]. To some extent, this debate has revolved around the methods used by two different research teams [80][81][82] An alternative hypothesis to the requirement of this HPTM for CENP-A centromeric incorporation states that its recruitment depends exclusively on residues located in the CATD, which comprises amino acids 75-114 [83]. However, only future research will clarify whether CENP-A residues that have recently drawn attention, along with their respective PTMs, play an essential part in the correct CENP-A deposition and centromeric function.
CENP-A methionine 1 is excised by methionine aminopeptidase, similarly to H3 (the amino acid count is displaced, leaving glycine 2 in position 1). This modification leaves glycine 1 exposed, resulting in trimethylation by NMRT [74,84]. Although CENP-A-G1me3 can be detected in the prenucleosomal pool, it is more abundant in nucleosomal CENP-A. Furthermore, this HPTM is required to recruit CENP-I and CENP-T and, hence, correct kinetochore function [84].
On the other hand, Cse4, the CENP-A homolog in S. cerevisiae, has been shown to suffer sumoylation, mediated by the E3 SUMO ligases Siz1 and Siz2 [85,86]. Recent work has demonstrated that the sumoylation of lysines 215 and 216 increases both the centromeric and non-centromeric deposition of Cse4, since they promote its interaction with the histone chaperones CAF-1 and Scm3 (Scm3 is the yeast homolog of human HJURP) [85,87]. Furthermore, replacing Cse4 K215 and 216 for arginines or alanines leads to chromosome mis-segregation [87]. Cse4 is also sumoylated on lysine 65, as a mechanism to prevent its incorrect localization [88]. This modification is recognized by the ubiquitin E3 ligases Slx5 and Psh1, which target mislocalized or overexpressed Cenh4 for proteasomemediated degradation [85,88]. Interestingly, Slx5 has a mammalian ortholog named RNF4, which is associated with the regulation of chromosome segregation in human cells [88,89]. Therefore, it will be interesting to determine whether mammalian CENP-A function is also regulated by sumoylation systems, similar to yeast Cse4.
Taken together, the experimental data show that CENP-A HPTMs are essential for proper chromosome segregation since they have essential roles in promoting CENP-A loading, restricting its deposition along the cell cycle, and correct kinetochore assembly. Nevertheless, the elucidation of the exact function of some of these marks will require further research. Moreover, the data generated by the ongoing controversies regarding the relevance of CENP-A PTMs outside of the CATD underscore the sensitivity of the CENP-A incorporation control systems to experimental manipulation, as well as the importance of comparative biology (such as in the case of Ser68) in the field. These aspects should be taken into consideration in future experimental settings.

HPTMs on SAC Activity
We reviewed that HPTMs result in essential modifications in the centromere structure and kinetochore function. Therefore, these modifications are expected to have implications for other downstream mechanisms involved in cell division. One is the spindle assembly checkpoint (SAC). This mechanism prevents chromosome mis-segregation and aneuploidy in mitotic cells by delaying anaphase until all chromosomes are attached to the mitotic spindle and the tension generated by poleward forces in these attachments is correct [90].
One HPTM that has been implicated in the regulation of SAC is the methylation of H3K4. It acts as a negative regulator of SAC through direct binding to the closed conformation of the mitotic arrest deficient protein 2 (C-Mad2), an important player of SAC that by interacting with Cdc20, prevents the activation of APC (anaphase promoter complex). Schibler et al. suggested that the binding to H3K4me could limit C-Mad2 interactions with Cdc20, as part of the normal regulation of SAC. On the contrary, the loss of H3K4me affected Mad2 localization in dividing cells, which in turn conferred resistance to benomyl in Saccharomyces cerevisiae due to a missing functional SAC [91]. Accordingly, Beilharz et al. also found that a loss of H3K4 methylation caused benomyl resistance in several strains of Saccharomyces cerevisiae and that this might be related to an increased expression of alpha tubulin, which in turn could enhance microtubule stability or spindle formation [92]. The authors suggested that since the active benomyl component MBC binds to microtubules, increased tubulin levels may directly contribute to benomyl resistance. Alternatively, another possibility is that abnormally enhanced microtubule stability could affect the tension in the mitotic spindle. The lack of tension generated by poleward forces in the spindle activates the SAC to prevent chromosome mis-segregation in the mitotic cell. In this regard, Luo et al. found that a small region of H3, K37-G44, is necessary to interact with and recruit Shugoshin (SGO1) to the pericentromeric region in Saccharomyces cerevisiae. Since SGO1 plays a key role in mitotic tension sensing, this interaction is essential to activate the SAC under a lack of tension in the mitotic spindle. The authors do not rule out the possibility of a post-translational modification of H3 responsible for the interaction with SGO1, but further exploration is needed [93]. What remains to be seen, either in yeast or mammal cells, is whether any specific methylated state of H3K4 could inhibit SAC by interacting with C-Mad2 or promote SAC by recruiting SGo1 to sense the tension between kinetochores and the spindle. Nonetheless, the evidence presented here uncovers an interesting regulating role of an HPTM in the SAC activity and the tension sensing in the mitotic spindle, highlighting the importance of H3K4me in preventing chromosome mis-segregation and possibly chromosome instability.

Environmental Factors, Histone Post-Translational Modifications, and Genome Maintenance
We reviewed that certain HPTMs play an essential role in proper chromosome condensation and centromere function during mitosis. Therefore, the abnormal alteration of these HPTMs or their regulators brings upon the cell severe consequences that can, in some cases, lead to genomic instability. Moreover, it is known that HPTMs and histone variants are regulated by intra and extracellular factors and by environmental factors. Consequently, the environment is also implicated in chromatin organization, gene expression, chromosome segregation, and a rise in genomic aberrations [94]. Here, we list some environmental factors that can modify histone marks related to chromosome structures important in mitosis.
Arsenic is a pollutant metalloid widely distributed in nature and ubiquitous in soil, water, and air. Humans are chronically exposed to arsenic compounds through contaminated food and drinking water [95,96]. In nature, it can be found in organic or inorganic form, with the latter being the most toxic. The metabolism of inorganic arsenic gives rise to different methylated forms, of which dimethylarsinous acid (DMAIII) and monomethylarsonous acid (MMAIII) are the most toxic in vitro studies [97,98]. Arsenic has multiple cellular effects; it inhibits DNA repair, increases cellular oxidative stress, promotes apoptosis, provokes mitotic alterations, and modifies epigenetic marks [99]. It has been shown that arsenic causes an increase in mitosis length, the formation of chromosomal lags, and the generation of aneuploidies in vitro [95,100,101]. The consequences of cellular exposure to arsenic depend on its chemical species and dose, e.g., sodium arsenite in high doses (greater than 5 micromolar) causes cell death after mitotic arrest instead of only delaying mitosis [95,101].
Although some of the effects of arsenic in mitosis have been associated with its binding to tubulin [102], the formation of the mitotic spindle [103], and the inhibition of proteins such as Plk1 [95], changes in histone marks could alter the mechanisms of condensation and centromere functioning, provoking the mitotic alterations observed in arsenic-treated cells. For example, Suzuki et al. showed that treatment with DMAIII results in the increased phosphorylation of H3S10, a mark associated with chromosome condensation. Furthermore, treatment with DMAIII affected the CPC's proper location and chromosome segregation [104]. Thus, alteration of this histone mark could inhibit adequate chromosome condensation and segregation. As previously mentioned, the phosphorylation of H3S10 allows the recruitment of Hst2p, which deacetylates the H4K16 residue by promoting chromosome compaction. Arsenic treatment decreases the overall levels of histone acetylation [105][106][107] and H4K16ac by binding to and inhibiting hMOF (the histone acetylase enzyme responsible for the acetylation of H4K16) [106,108]. Because this alteration was evaluated in the interphase, it will be relevant to determine whether arsenic also modifies the acetylation of H4K16 in mitosis and the consequences it may have on chromosome segregation. As already stated, the presence of H3K4me2 and H2Bub1 modifications are significant for CENP-A recruitment and centromere maintenance. In this regard, it has been shown that cellular exposure to arsenic causes an increase in global H3K4me2 and H3K4me3 [109][110][111]. Although the centromeric region has a significant level of H3K4me2, it also has low levels of H3K4me3, so the increase in the latter mark could modify the structure of centromeric chromatin and alter the recruitment of CENP-A.
On the other hand, treatment with arsenic for long periods has the opposite effect, causing a decrease in both H3K4me2 and H3K4me3, the latter being associated with an increase in KDM5 (a histone demethylase) [112]. In C. elegans, it was shown that arsenic treatment promotes a transgenerational reduction in H3K2me2 levels associated with a decrease in spr-5 expression (which codes for a histone demethylase that removes the H3K4me2 mark) [109]. On the other hand, arsenic can bind to the RNF20 protein (the enzyme that generates histone H2B ubiquitination) and inhibit its function, causing a decrease in H2Bub1 levels [113]. As the overall modification of H3K4me2 and H2Bub1 by exposure to arsenic has been evaluated, it will be relevant to define whether these alterations are also present at the centromeric level. It is also essential to determine whether these changes are responsible for specific defects during chromosome segregation.
Nickel (Ni) is a metal found in the environment and originated from the erosion of rocks and volcanic origin. Most of the Ni has an anthropogenic origin due to fossil fuels and industrial processes [114]. Ni is deposited in the soil and is also found in the atmosphere. It can be found as elemental nickel (Ni), nickel oxide (NiO), nickel chloride (NiCl2), and nickel sulfate (NiSO4), among others [115]. Different studies have shown that exposure to Ni causes alterations in chromosome segregation. The treatment of cell cultures with Ni promotes an increase in cells with kinetochore-positive micronuclei [115][116][117]. Moreover, an increase in aneuploid cells and alterations during anaphase have been found after treatment with Ni compounds [116,118].
In various models, it has been shown that Ni can modify histone marks, some of which may be related to mitotic chromosome formation and functioning. In vitro assays have demonstrated that Ni promotes chromatin condensation independently of the cellular machinery. Moreover, Ni treatment is associated with DNA methylation and gene silencing [119][120][121]. As previously mentioned, H3S10ph is an important mark for chromosome compaction. Cells treated with Ni increase the overall levels of H3S10ph [122]. However, the increase in H3S10ph was observed only at the interphase, so it is unclear whether this alteration can affect mitotic chromosome compaction. The role of H3S10ph in the compaction of chromosomes is also associated with the deacetylation of histone H4. Ni diminishes global acetylation of H4 [123,124]. It has been proposed that the inhibition of H4ac is due to the formation of ROS caused by Ni [125]. However, it has also been associated with the direct binding of Ni to histone H4, which prevents its binding to HATs [126]. Moreover, Ni has been shown to decrease local acetylation of H3 and increase H3K9me2 and H3K9me [127,128]. Finally, Ni also increases global levels of H2Aub and H2Bub by inhibiting the deubiquitination of both histones [129]. In most of these studies, changes in histone marks have been studied globally, so it will be relevant to define whether these alterations are present in specific loci and if Ni alters particular marks. It will also be essential to know if modifications in histone marks are associated with defects in segregation caused by Ni and if these marks are specifically modified during mitosis.
In addition to Ni, other metals can modify histone marks and chromosome segregation. Cadmium, for example, causes the formation of aneuploidies in vitro and changes the methylation patterns of DNA and histones [130]. Cadmium also increases H3K4me3 and H3K9Me2 by inhibiting the demethylases that remove these marks [131].
On the other hand, ambient particulate matter (PM) can modify both histone marks and chromosome segregation. Exposure to soil and road dust decreased the expression of Suv39h1 (a histone methyltransferase responsible for H3K9 methylation) with a concomitant increase in centromeric satellite DNA expression. As previously mentioned, the expression of these sequences is tightly associated with CENP-A incorporation. Accordingly, the authors of this work observed chromosome aberrations under soil dust exposure in vitro [132]. Airborne particulate matter also modifies histone mark patterns. PM10 (particles with an aerodynamic diameter smaller than 10 µm) exposure promotes acetylation of histone H4 by increasing HAT activity [133]. Moreover, PM10-exposed human cells showed an increase in micronucleated and trinucleated cells [134]. However, particles obtained from different regions or different times of the year may have a different composition. The same PM10 sample must be used for experiments to determine whether PM10 exposure can alter histone marks and chromosome segregation.

Conclusions
HPTMs and the deposition of histone variants play a crucial role in distinct stages of mitosis, starting from chromatin condensation, centromere formation, kinetochore assembly, and dynamics of the mitotic spindle during cell division. Therefore, the deregulation of HPTMs can lead to chromosome mis-segregation and errors in cell division. It has been suggested that the cell responds to environmental stimuli and challenges through HPTMs and the deposition of histone variants [135]. Therefore, it is expected that chromatin condensation and mitosis regulation might be disturbed by environmental insults. Exposure to genotoxic and radiation agents is related to changes in certain HPTMs and the deposition of histone variants and is associated with deleterious effects such as DNA damage, aneuploid-associated syndromes, and chromosome instability, a well-known hallmark of cancer. It has been suggested that certain environmental factors are associated with cancer, but only a few examples are well explained, and their carcinogenic mechanisms are known in detail [135,136]. Plenty of pollutant agents are widely spread in our environment due to the current human lifestyle and industrial and transport activities. Their impact on distinct cell functions and pathways needs to be addressed. The deregulation of mitotic HPTMs will certainly uncover the carcinogenic potential of many of those pollutant agents and help societies and decision-makers to search for healthier and safer alternatives.